Abstract
BACKGROUND:
Studying the pleiotropic effects of medicines can reveal their unexplored therapeutic advantages. It promotes repurposing established drugs or identifying compounds that affect multiple targets. The widely prescribed antihypertensive agents, ramipril and olmesartan, because of their ability to reduce C-reactive protein activity, may also modulate the immune system.
AIM:
The study aims to demonstrate the potential immunomodulatory effects of ramipril and olmesartan, which would be beneficial for the management of inflammation in patients with coexisting hypertension.
MATERIALS AND METHODS:
The anti-inflammatory properties of the drugs were studied in acute and chronic models of inflammation. The immunomodulatory action was evaluated using a neutrophil adhesion test, proinflammatory and anti-inflammatory cytokine assay, and hemagglutination antibody titer assay. Histopathological analyses of rat paw edema were also performed.
RESULTS:
Both drugs demonstrated enhanced neutrophil adhesion, increased hemagglutination antibody titers, reduced paw edema, and improvement in cellular and structural changes associated with inflammation. Drug treatment also inhibited proinflammatory cytokine production while promoting anti-inflammatory cytokine production.
CONCLUSION:
The results indicate the immunomodulatory potential of ramipril and olmesartan at equipotent antihypertensive doses, compared with the standard drugs, in Wistar albino rats. The results reveal that olmesartan exhibits superior immunomodulatory properties compared with Ramipril.
Keywords: Anti-inflammatory, histopathology, immunomodulation, pleiotropic effects
Introduction
The principle of “one drug, one target, and one disease” has led to the successful development of numerous medications for specific illnesses. However, drug discovery faces significant challenges due to the complexity of diseases, which often involve abnormalities in cellular structure and function, necessitating innovative approaches to develop effective treatments. Repurposing existing drugs to modulate multiple targets in a pathogenic cascade provides a more effective treatment strategy, leveraging known safety profiles and mechanisms of action.
Inflammation is a crucial physiological response to harmful invaders and injury. Acute inflammation, a short-term process characterized by rubor, calor, tumor, and dolor, helps to eliminate pathogens and initiate healing. However, chronic inflammation is more extended and can result in serious health issues, notably cardiovascular disorders, cancer, and autoimmune disorders. Balancing acute and chronic inflammation is vital for overall health.[1] In addition, controlling persistent low-grade inflammation helps reduce blood vessel damage.
C-reactive protein (CRP) is a vital marker of inflammation. Elevated serum CRP levels are associated with an increased risk of cardiovascular diseases, autoimmune diseases, and inflammatory conditions, such as rheumatoid arthritis, as well as certain cancers.[2] Apparently, a reduction in CRP levels during treatment indicates therapeutic efficacy.
Several pharmacological agents reduce CRP levels in blood plasma, including lipid-lowering agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and antidiabetic agents.[3] Among these CRP-reducing drugs, we investigated ramipril (an ACE Inhibitor) and olmesartan (an ARB) for their adjuvant role in treating inflammation that occurs with coexisting hypertension. Ramipril inhibits the ACE, which converts angiotensin I to angiotensin II, which can constrict blood vessels. Reduced levels of angiotensin II lower the blood pressure by dilating the blood vessels.[4] Olmesartan reduces the action of the hormone angiotensin II. ARBs specifically block the hormone’s action by targeting its receptors, Angiotensin II type 1 receptors in the heart, kidneys, and blood vessels. Blocking angiotensin II activity also helps reduce blood pressure.[5]
Materials and Methods
Test drugs
Ramipril (0.9 mg/kg body weight),[6] olmesartan (0.5 mg/kg body weight),[7] reference standard immunomodulatory drug levamisole (2.5 mg/kg body weight),[8] and reference standard anti-inflammatory drug aspirin (100 mg/kg body weight) were procured in pure powdered form from Yarrow Chem Chemicals, Mumbai, India. The vehicle used was normal saline. All other chemicals used were of analytical reagent grade and prepared aseptically.
Experimental animals
Male Wistar albino rats (150–250 g) were obtained from Kerala Veterinary and Animal Sciences University, Kerala, India. They were maintained in the animal house under standard conditions and were fed a standard pellet diet and water ad libitum. The paddy husk bedding was replaced daily to maintain a clean and dry environment for the animal. Animal experiments were conducted with the prior approval of the Institutional Animal Ethics Committee (No. 22122017-6 dated November 01, 2021). The maintenance of rats and experimental protocols used were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, India.
Neutrophil adhesion test
The Wistar albino rats were first categorized into four groups, each comprising six animals (n = 6). Group 1 (Control): isotonic saline solution (500 µl/day, orally), Group 2 is the standard reference drug group, levamisole (2.5 mg/kg bodyweight, orally), Group 3 (Test 1): ramipril (0.9 mg/kg bodyweight), Group 4 (Test 2): olmesartan (0.5 mg/kg bodyweight). Animals in each group received oral drug treatments for 10 days, and on the 11th day, blood samples were collected from the retroorbital plexus of each rat. Blood was collected in ethylenediaminetetraacetic acid (EDTA) vials and analyzed for total leucocyte count (TLC) and differential leukocyte count (DLC).[9] TLC and DLC were both performed using EDTA blood. An updated Neubauer Chamber was used for TLC, and the analysis was performed manually using the hemocytometer technique. A white blood cells pipette was used to draw blood up to the 0.5 mark. Once the initial cell counts were obtained, the blood samples were exposed to nylon fiber (80 mg/ml) and incubated for 15 min at 37°C. Postincubation, TLC and DLC were reassessed. The neutrophil index (NI) was calculated as the product of the TLC and the percentage of neutrophils in the blood samples. Percent neutrophil adhesion was calculated using the formula,
NIu is the NI of untreated samples with nylon fiber; NIt is the NI after incubation with nylon fiber.[10]
Indirect hemagglutination antibody titer
Antigen preparation
Sheep blood was collected from a slaughterhouse in Alsever’s Solution (1:1) under aseptic conditions and kept immediately at 4°C. Sheep red blood cells (SRBCs) for immunization were prepared by centrifugation at 2000 RCF for 10 min. Then, the supernatant was removed, and the pellet was washed 4 times with physiological saline (0.9% w/v NaCl). After washing, the SRBC were suspended in phosphate-buffered saline (PBS) (pH 7.2). Alsever’s solution contained 2.05% dextrose, 0.8% sodium citrate, 0.55% citric acid, and 0.44% sodium chloride, with a pH range of 6–7.
After 10 days of drug treatment, the rats were intraperitoneally immunized with 0.2 ml of SRBCs (10%). On the 5th day after immunization, blood was collected from each rat through the retro-orbital plexus, and the serum was separated. The serum was incubated at 56°C for 30 min for complement inactivation. Then, two-fold serial serum dilutions were prepared in 100 µl of PBS (pH 7.2) in 96-well microtiter plates (Himedia, India). Then, 100 µL of a 1% SRBC suspension in PBS was added. After proper mixing, the plates were incubated at room temperature for 2 h and visually examined for agglutination. The antibody titer was determined as the highest serum dilution that showed visible hemagglutination.[11]
Assessment of acute inflammation using carrageenan-induced rat paw edema
Anti-inflammatory properties were studied in the second set of experiments. The grouping of animals and the administration of drugs were mentioned in the first experiment. A standard dose of 100 mg/kg aspirin was administered as the reference treatment. After an hour, 0.1 ml of 1% carrageenan was injected beneath the right rear paw. The paw volume was measured promptly using a Digimatic digital micrometer (Mitutoyo American Corporation) at 0, 1, 2, 3, and 4 h; the average paw volume in each group was recorded using a micrometer.[12] To assess antiinflammatory activity, the percentage decrease in swelling was measured in rats treated with ramipril and olmesartan and compared with rats receiving only carrageenan. The inhibition percentage (%) was calculated using the formula,
Vc is the paw volume in control, Vt is the paw volume in the test.
Histopathological analysis
Upon completing the paw edema test, rats were euthanized, and paw tissues were taken for histopathological processing. Tissues fixed in 10% neutral buffered formalin were dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Sections were cut, mounted, deparaffinized, rehydrated, and stained with hematoxylin and eosin. Then, it was visualized under × 40 magnification.[13]
Formalin-induced chronic paw edema assay
The formalin-induced paw edema technique is employed to measure chronic inflammatory response. The experimental medications explained above were administered via oral gavage daily for ten consecutive days, adhering to exact 24-h dose intervals. 0.1 ml of 2% formalin is subcutaneously injected into the right hind paw of rats on day 5 of the experiment. The relative paw thickness was assessed using a digital micrometer 1 h before formalin injection and monitored continuously for 5 days postinjection. The same formula employed in the carrageenan model was utilized here.[14]
Analysis of cytokines
Proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) and the anti-inflammatory cytokine IL-10 were quantified using an indirect enzyme-linked immunosorbent assay, as described by Surendran et al.[15] Serum samples (100 μL) were incubated overnight at 37°C in 96-well plates. After washing with PBS, a blocking buffer (200 μL of 0.2% gelatin in 0.05% Tween 20; Merck, Germany) was added and incubated for 1 h. Diluted primary antibodies (1:500 dilution) for IL-6 (Cat: Sc-57315, Santacruz, USA), TNF-alpha (Cat Sc-52746, Santacruz, USA), and IL-10 (Cat: Sc-53308, Santacruz, USA) were added and incubated for 2 h, followed by washes with PBS Tween. A secondary HRP-conjugated antibody (Santacruz, USA) (1:500) was added and reacted for 1 h. O-dianizidine hydrochloride (200 μL) was added to facilitate color development, and the reaction was halted with 50 μL of 5N HCl. Absorbance was measured at 415 nm. Protein concentration was determined using the Lowry method.[16] All assays were carried out as per the bioanalytical guidelines provided by the manufacturer (Santa Cruz Biotechnology, USA). Samples were run in triplets, and positive and negative controls ensured assay performance. Interassay variability was observed below 10%.
Statistical analysis
The results from six animals are expressed as mean ± standard error Statistical significance was analyzed using one-way ANOVA followed by Tukey’s multiple-range test in SPSS Ver. 20 (SPSS Inc. Chicago, IL, USA). The criterion considered for statistical significance was P ≤ 0.05.
Results
Effect of ramipril and olmesartan on neutrophil adhesion test
The neutrophil adhesion percentages were significantly higher (P < 0.05) in both drug-treated groups [Figure 1]. Olmesartan (27.7%) showed greater activity than ramipril (24.3%).
Figure 1.
Neutrophil Index in the presence of Ramipril and Olmesartan. Total leukocyte count and differential leukocyte count were assessed to calculate neutrophil index in rats after 10-days oral drug administration. Oral saline administered group was taken as the negative control. Neutrophil adhesion was determined post incubation with nylon fiber. NIU = Neutrophil index of nylon-untreated groups, NIT = Neutrophil index of nylon-treated group. Values are represented as mean ± standard error (n = 6). Means with the same letter are not significantly different by Tukey’s multiple range tests (P ≤ 0.05)
Effect of ramipril and olmesartan on indirect hemagglutination antibody titer
The hemagglutinating antibody titer was higher in the treated groups than in the control [Figure 2]. The Olmesartan-treated group showed a marked rise in titer value compared to the control group.
Figure 2.
Indirect Hemagglutination antibody titer: After 10-day’s drug treatment, rats were immunized with sheep red blood cells (SRBC) suspension, serum was collected after 5 days and mixed with 1% SRBC suspension for assessing agglutination. Negative control was saline treated group. Values are represented as mean ± standard error of three experiments. Means with the same letter are not significantly different by Tukey’s multiple range tests (P ≤ 0.05)
Effect of ramipril and olmesartan on acute inflammation
The ramipril group exhibited a notable percentage suppression of paw edema (46.38%), as did the olmesartan group (46.81%), in comparison to the control group (51.06%) (P < 0.05) [Figure 3].
Figure 3.
The effect of Ramipril and Olmesartan in acute inflammation: Paw volume of the drug-administered rats was measured before and after carrageenan injection and compared to the reference standard aspirin. Values are represented as mean ± standard error of six animals in each group, *P < 0.05
Histopathological observations
Normal rats exhibited the typical histological architecture of both the epidermis and dermis. In the carrageenan and formalin control groups, hyperkeratinization and infiltration of inflammatory cells were noted. Sections from the rats administered ramipril and olmesartan displayed a marked reduction in histological alterations, with decreased keratinization and a normal appearance of the dermis and hypodermis [Figure 4].
Figure 4.
Histology of the rat paw tissue (H and E) after the development of carrageenan-induced paw edema in × 40. Untreated control (a) showing normal histology. Carrageenan-alone treated group (b) show marked inflammatory reactions in the dermis with intramuscular infiltration of massive number of neutrophils while Ramipril treated rats (c) and Olmesartan treated group (d) show weak inflammatory reactions
Effect of ramipril and olmesartan on chronic inflammation
In the present study, aspirin at a dosage of 100 mg/kg exhibited a percentage inhibition of 50.56%; ramipril at 0.9 mg/kg, 46.33%; and olmesartan at 0.5 mg/kg, 46.5% [Figure 5].
Figure 5.
The effect of Ramipril and Olmesartan in formalin-induced paw edema. After administering the drugs via oral gavage for ten days, paw thickness of Wistar rats was measured using a Vernier Caliper before and after a formalin injection on day 5. Values are represented as mean ± standard error of six animals in each group, *P < 0.05
Effect of ramipril and olmesartan on cytokine production
Ramipril and olmesartan inhibited the release of proinflammatory cytokines IL-6 and TNF-α. The anti-inflammatory cytokine, IL-10, was significantly increased after the drug treatments [Figure 6].
Figure 6.
The effect of Ramipril and Olmesartan on the release of cytokines. After 10 days of oral drug administration, levels of interleukin 6 (IL 6), tumor necrosis factor alpha and IL 10 were measured by Indirect Elisa. Values represent mean ± standard error of three independent experiments, analysed by one-way ANOVA followed by Tukey’s test, P < 0.05 were considered significant. Values carrying same alphabet did not vary significantly from each other
Discussion
In recent years, the use of multitarget drugs has attracted significant attention as a promising therapeutic strategy for diseases with complex etiologies. Employing multitarget medicines can provide improved pharmacokinetic profiles, better patient outcomes, reduced risk of adverse drug reactions, and a lower financial burden. Chronic inflammation is a central mechanism underlying the pathogenesis of cardiovascular disorders, neurodegenerative conditions, and autoimmune syndromes. Polypharmacy is not advocated in chronic disease management due to increased risk of adverse drug events.[17] By assessing the immunomodulatory effects of Ramipril and Olmesartan in validated animal models, this study provides preclinical evidence supporting their potential repurposing for inflammation-associated disorders. Employing equipotent antihypertensive doses of ramipril (0.9 mg/kg) and olmesartan (0.5 mg/kg) enabled a direct head-to-head assessment of their immunomodulatory effects, clarifying how each agent might influence immune-mediated pathology in hypertensive patients. These doses were selected to achieve maximal receptor occupancy and clinically relevant pharmacodynamic activity while avoiding nonspecific toxicity.[18] This also enables assessment of immunological effects independent of dose-related confounding factors, potentially guiding clinicians towards agents with favorable immunomodulatory profiles.
Neutrophils serve as the primary phagocytic responders to pathogenic invasion, and the neutrophil adhesion assay is widely used to assess neutrophil margination within the vasculature as a marker of immune activation. The inclusion of Group 1 (receiving only isotonic saline) serves as a control group, representing the physiological state without drug intervention. This group provides a baseline reference for comparison and accounts for natural variability and procedural influences along with the experimental groups. By comparing the outcomes, any observed differences can be attributed to the drugs’ pharmacological actions. Neutrophil adherence, as shown by ramipril (24.3%), may be due to its effects on inhibiting the conversion of angiotensin I to angiotensin II, resulting in a downstream reduction of proinflammatory cytokines and adhesion molecules, which may, in turn, diminish neutrophil adhesion to the vascular endothelium. Conversely, olmesartan attenuates neutrophil adhesion by (27.7%) by selectively blocking angiotensin II type 1 receptors, thereby suppressing the proinflammatory signaling cascade and reducing the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.[19] Collectively, these mechanisms highlight the distinct immunomodulatory roles of these pharmacological agents in regulating neutrophil-endothelial interactions, with olmesartan exhibiting a more pronounced effect.
Cytokines play a crucial role in differentiating lymphocytes, regulating inflammation, promoting cell survival, defending against microbial infections, and mediating apoptosis and other immunological responses. Both ramipril and olmesartan significantly inhibited TNF-α (P = 0.021) and IL-6 (P = 0.006) production, supporting their potential role in managing chronic inflammation. In addition, the statistically significant increase in IL-10 levels following treatment (P = 0.026) highlights its potential as an anti-inflammatory agent. IL-10 regulates proinflammatory cytokine production and promotes immune cell proliferation. This dual action of decreasing TNF-α and IL-6, and increasing IL-10, suggests that these agents can shift the immune response toward a more anti-inflammatory state. These findings reinforce their therapeutic potential for inflammatory conditions.[20]
In indirect hemagglutination antibody titer, test animals classified as naïve are generally considered immunologically unexposed to SRBC, indicating that antibodies detected after immunization primarily reflect treatment-induced immune responses. Comparing antibody titers between treated and untreated groups allows assessment of immunomodulatory effects relative to controls.[21] The enhanced antibody titres (P = 0.021) in response to SRBC suggest a stimulatory effect on humoral immunity, potentially mediated through effects on B-cell function.
Carrageenan triggers TNF-α release, inducing IL-1 β and IL-6 production, which, in turn, upregulate COX-2 expression and promote the production of proinflammatory prostaglandins.[22] Neutrophil infiltration also plays a crucial role in carrageenan-induced paw inflammation. In our study, both drugs significantly reduced neutrophil infiltration in the treated paws, as shown by histopathological analyses. Formalin is yet another inflammatory agent,[23] and assessment of formalin-induced chronic inflammation reveals three phases: the transudative phase (first 3 h), the exudative phase (3–72 h), and the proliferative phase (3–6 days). The initial phase involves the direct activation of sensory neurons and the release of neurotransmitters, whereas the late phase triggers additional inflammatory responses and cytokine release.[24] Ramipril (46.3%) and Olmesartan (46.5%) reduced paw swelling by inhibiting proinflammatory mediators and modulating inflammatory pathways. Histological analysis of paw samples from the carrageenan-only-treated group revealed focal infiltration of inflammatory cells, primarily neutrophils, indicating disruption of the inflammatory system. Thus, treatment with ramipril and olmesartan significantly reduced inflammatory cell infiltration and edema.[25,26,27]
ACE inhibitors and ARBs may confer neuroprotective effects in neurodegenerative diseases by reducing oxidative stress, modulating neuroinflammatory cytokines, and preserving blood–brain barrier integrity, suggesting potential therapeutic relevance for delaying or modifying disease progression.[28] Both agents exhibited anti-inflammatory effects in joint tissues, reducing synovial inflammation, improving mobility, and slowing disease progression, making them potential adjuncts for patients with hypertension and inflammatory arthropathies.[29] In the post-COVID-19 setting, they may also mitigate residual inflammation, protect cardiopulmonary function, and reduce long-term complications of postacute sequelae of SARS-CoV-2 infection, extending their utility in postinfectious inflammatory syndromes.[30] All these data highlight the anti-inflammatory effects of ramipril and olmesartan, which act as a unifying driving mechanism, benefiting across multisystem disease management.
Among the two drugs studied, this study demonstrates that olmesartan exhibits superior immunomodulatory properties compared to ramipril. For example, in the neutrophil adhesion assay, olmesartan showed a substantial % activity rate of 27.7%, whereas ramipril lagged at 24.3%. Similarly, results from the hemagglutination titer assay revealed that olmesartan’s activity was nearly comparable to that of the control drug, in stark contrast to ramipril, which exhibited only minimal activity.
Conclusion
Building on existing evidence, these findings are anticipated to offer valuable insights into the pleiotropic effects of both drugs. Further research, including randomized controlled trials and real-world observational studies, is necessary to understand the scope of these pleiotropic effects and to identify the patient populations most likely to benefit.
Data availability statement
All data pertaining to the study are included in the manuscript.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data pertaining to the study are included in the manuscript.